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Evaluation of Transdermal Absorption of Ketoprofen in a Rabbit Model Pharm.D. Candidates 2014: Eliza S. Daubert, Christina Inteso, Manuel Isherwood, Trey Tietz
Arthur H. Kibbe Ph.D., Adam L. VanWert Pharm.D., Ph.D. Department of Pharmaceutical Sciences, School of Pharmacy, Wilkes University, Wilkes-Barre, PA
Materials & Methods (continued) Introduction
Ketoprofen, R,S- 2-(3-Benzoylphenyl) proprionic
acid, is a traditional non-steroidal anti-inflammatory
drug (NSAID) commonly administered orally as an
anti-inflammatory agent, analgesic, and anti-pyretic. It
exerts its action by inhibiting the COX-1 and COX-2
enzymes which mediate the formation of prostaglandin
precursors and thus controls pain, inflammation, and
fever in the body[1,2]. As an oral preparation,
absorption of ketoprofen is almost complete, making
this type of therapy very effective[3]. A known adverse
effect of this administration route, however, is
gastrointestinal irritation and ulceration due to the local
effects of prostaglandin synthesis suppression in the
gastric mucosa as the drug undergoes absorption[1].
Also, a number of patients are not good candidates for
oral therapy due to age, disease state, or other
factors.
A potential drug delivery system that would
overcome these problems is a topical preparation
such as a gel. Based on the physiochemical and
pharmacokinetic properties of this NSAID, transdermal
absorption of ketoprofen is possible[4]. The primary
purpose of this study was to assess the extent to
which this drug can be absorbed transdermally from a
gel preparation and to analyze whether therapeutic
drug levels can be achieved to provide systemic anti-
inflammatory, analgesic, and anti-pyretic effects. The
secondary purpose was to evaluate the rabbit as a
useful animal model to test differences in formulation
on the amount of drug absorbed from the site of
administration.
Figure 2: Ketoprofen Levels in Rabbits
The results showing peaks of the ketoprofen
plasma samples were not significantly higher than those
of the blank baseline indicate that significant systemic
absorption of ketoprofen from a transdermal gel
preparation could not be detected. Additionally, the trace
levels observed were transient and variable. This finding
suggests further studies are needed to evaluate higher
doses of compounded ketoprofen transdermal gels. The
results of this study also bring into question the
usefulness of a rabbit model to test potential
transdermal absorption of ketoprofen in vivo due to
significant metabolic and physiological differences from
a human model. Future studies should consider a
different assay to detect ketoprofen plasma levels.
Printed by
Results (continued)
Discussion/Conclusions
The mobile phase was delivered at a rate of 1.2 mL/min and time between injections was 10 minutes[5].
Solutions of 0.5 μg/μL, 5 μg/μL, 10 μg/μL, 100 μg/μL and 1 mg/μL ketoprofen in acetonitrile were prepared and
used to construct a standard curve.
Ketoprofen was formulated into two commonly used transdermal preparations as shown in Figure 1, below:
Figure 1: Ketoprofen Gel Formulations
The Institutional Animal Care and Use Committee (IACUC) approved the study protocol. The four rabbits were
housed in a normal 12 hour light/dark environment with water freely available and were fed 150 grams of standard
rabbit chow daily in addition to grass. To carry out this study, a given animal was restrained in a rabbit stock and
the inside of one ear was dosed with 0.2 mL of 1 percent ketoprofen gel (1 mg/kg). Blood was collected in
heparinized tubes from the marginal veins of the opposite ear at time intervals of 0, 0.5, 1, 1.5, and 2 hours. The plasma was separated via centrifugation at 2000 g for 2 minutes and stored at 5 ⁰C for further analysis.
The extraction procedure utilized a sample of 100 μL of plasma to which 400 μL acetonitrile was added[5]. This
was vortex mixed for 2 minutes and centrifuged at 21000 g for 5 minutes. The clear supernatant was utilized for
reversed phase high performance liquid chromatography (HPLC) analysis. After establishing a steady baseline, the
standard and plasma samples were injected and the resulting chromatogram recorded. Linear regression of the
standard curve data was calculated by plotting the peak area against the drug concentration in micrograms per
millimeter.
The first formulation used Pluronic-Lecithin-Organogel (PLO) as a base[6]:
The organic phase, composed of Lecithin, was made by the general formula below:
Preparation of Lecithin Solution
Lecithin Soya Granular 100 gm
Sorbic Acid NF 660 mg
Isopropyl Palmitate 100 gm
The aqueous phase, composed of a Pluronic 127 gel, was made using the following formula:
Preparation of Pluronic 127 NF 20% Solution
Pluronic 127 NF 20 gm
Potassium Sorbate 300 mg
Distilled Water 100 mL QS
Ketoprofen PLO gel was made using the following formula:
Preparation of 1% Ketoprofen PLO Gel
Ketoprofen USP 30 mg
Lecithin (organic) solution 0.6 mL
Pluronic 127 (aqueous) solution 2.4 mL QS
The ketoprofen was dissolved in the lecithin solution before introducing the aqueous Pluronic phase then mixed well to form a
homogenous gel.
The second formulation used PCCA’s Lipoderm® base. The gel was made based on the following formula:
Preparation of 1% Ketoprofen Lipoderm TDG
Ketoprofen USP 30 mg PCCA Lipoderm® 3 mL QS
Propylene glycol 2 gtts Ketoprofen was dissolved in propylene glycol and mixed well with the Lipoderm® base.
Materials & Methods
Ketoprofen and all other materials needed to
compound the gel formulations were purchased from
Sigma-Aldrich. Lipoderm base was purchased from
the Professional Compounding Centers of America
(PCCA). Ammonium sulfate was obtained from Acros
Organics. HPLC grade acetonitrile was purchased
from Spectrum. Xylenes and HPLC grade methanol
were procured from Fisher Scientific. Four New
Zealand white rabbits approximately 1.5 kg each were
obtained from Charles River Laboratories.
The Water’s HPLC system included a 717 plus
autosampler, a 600 solvent delivery pump, a 486
tunable absorbance detector, and a Supelco 18-LC
analytical column. The chromatic separation was
performed through injection of 20 μL samples at room temperature (22 ⁰C) and detected at 265 nm. The
mobile phase consisted of 59% methanol, 26%
acetonitrile, and 15% water.
References:
1. Julou L, Guyonnet JC, Ducrot R et al. Ketoprofen (19.583 R.P.) (2-(3-benzoylphenyl)-propionic acid): main pharmacological properties—outline of toxicological and
pharmacokinetic data. Scand J Rheumatol. 1976; 14(Suppl): 33-44.
2. Simon LS, Mills JA. Nonsteroidal antiinflammatory drugs. N Engl J Med. 1980; 302: 1237-43.
3. Ketoprofen. In: Lexi-Comp Online™ [database on the Internet]. Hudson, OH: Lexi-Comp, Inc. [cited 2012 Feb. 1]. Available from: http://www.crlonline.com/crlonline
4. Beetge E, Du Plessis J, Muller DG, et al. The influence of the physiochemical characteristics and pharmacokinetic properties of selected NSAID’s on their transdermal
absorption. Int J Pharm. 193 (2000): 261-4.
5. Proniuk S, Lerkpulsawad S, Blanchard J. A simplified and rapid high-performance liquid chromatographic assay for ketoprofen in isopropyl myristate. J Chromatogr. Sci.
1998; 36: 495-8.
6. Hoffman SB, Yoder AR, Trepainer LA. Bioavailability of methimazole in a pluronic lecithin organogel (plo) in health cats. [Internet]. Oxford: Blackwell Scientific: 2002.
Available from: http://web.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=7b1275de-99cd-45b5-912c-1d8cd6c5b3e1%40sessionmgr14&vid=7&hid=12
Results
A standard curve of ketoprofen gave linear results with a correlation coefficient of 0.999 and a retention time of
5 minutes. Unfortunately, due to a confounding endogenous substance in the rabbit plasma, minimal baseline separation was observed. The Lipoderm® gel showed slightly higher peaks in comparison to the PLO gel formulation.
The results for the four rabbits, each treated with the same dose of ketoprofen in two different vehicles, appear in
Figure 2, to right.
Disclosure Statement
The authors of this presentation have the following to
disclose concerning possible financial or personal
relationships with the commercial entities that may have
direct or indirect interest in the subject matter of this
presentation:
Arthur H. Kibbe: Nothing to disclose
Adam L. VanWert: Nothing to disclose
Eliza S. Daubert: Nothing to disclose
Christina Inteso: Nothing to disclose
Manuel Isherwood: Nothing to disclose
Trey Tietz: Nothing to disclose